Transgenic mice overexpressing γ—aminobutyric acid transporter subtype I develop obesity

Transgenic mice ubiquitously overexpressing murine γ-aminobutyric acid transporter subtype I were created.Unexpectedly,these mice markedly exhibited heritable obesity,which features significantly increased body weight and fat deposition.Behavioral examination revealed that transgenic mice have slightly reduced spontaneous locomotive capacity and altered feeding pattern.This preliminary finding indicates that the inappropriate level of γ-aminobutyric acid transporters may be directly or indirectly involved in the pathogenic mechanism underlying certain types of obesity.     

Impaired reproduction in transgenic mice overexpressing γ-aminobutyric acid transporter Ⅰ (GAT1)

It is well documented that γ-aminobutyric acid (GABA) system existed in reproductive organs. Recent researches showed that GABA_A and GABA_B receptors were present in testis and sperm, and might mediate the acrosome reaction induced by GABA and progesterone. GABA transporter Ⅰ (GAT1) also existed in testis and sperm, but its physiological function was unknown. In the present study, we used GAT1 overexpressing mice to explore GAT1 function in male reproductive system. We found that the expression level of GAT1 continuously increased in wild-type mouse testis from 1 month to 2 months after birth. GAT1 overexpression in mouse affected testis development, which embodied reduced testis mass and slowed spermatogenesis in transgenic mice. Moreover, transgenic mice showed increase of the percentage of broken sperm. The further study revealed that the reproductive capacity was impaired in GAT1 overexpressing mice. In addition, testosterone level was significantly low in transgenic mice compared with that in wi     

Impaired reproduction in transgenic mice overexpressing γ-aminobutyric acid transporter I （GAT1）

It is well documented that 7-aminobutyric acid (GABA) system existed in reproductive organs. Recent researches showed that GABAA and GABAB receptors were present in testis and sperm, and might mediate the acrosome reaction induced by GABA and progesterone. GABA transporter I (GAT1) also existed in testis and sperm, but its physiological function was unknown. In the present study, we used GAT1 overexpressing mice to explore GAT1 function in male reproductive system. We found that the expression level of GAT1 continuously increased in wild-type mouse testis from 1 month to 2 months after birth. GAT1 overexpression in mouse affected testis development, which embodied reduced testis mass and slowed spermatogenesis in transgenic mice. Moreover, transgenic mice showed increase of the percentage of broken sperm. The further study revealed that the reproductive capacity was impaired in GAT1 overexpressing mice. In addition, testosterone level was significantly low in transgenic mice compared with that in wild-type mice. Our findings provided the first evidence that abnormal expression of GAT1 could result in dysgenesis,and indicated that GAT1 might be therapeutically targeted for contraception or dysgenesis treatment.     

Overexpression of γ-aminobutyric acid transporter subtype I leads to susceptibility to Kainic acid-induced seizure in transgenic mice

INTroDUCTION.7-aminobutyric acid (GABA), a major in-hibitory neurotransmitter in vertebrate brain, in--duces neuronal inhibition via GABA receptors. Adeficiency of GABAergic inhibition has been hy-pothesized to be a principal factor in the patho-genesis of epilepsy[1]. GABA transporters are im-portant components of the GABAergic system, andfunction in part to terminate the GABA transmis-sion through rapid re-uptake of GABA into thepresynaptic neurons and surrounding gliaJ cells[2…     

Ab-initio SCF investigation ofγ-aminobutyric acid

Summary The potential energy surface of the neutral form of-aminobutyric acid was investigated by means of ab-initio 4-31 G SCF calculations. Geometries, energies, and selected wave numbers of all 62 symmetry unique local minima are reported. Intramolecular interactions and all reactions, which involve the intramolecular hydrogen bond, are discussed and compared with those present in the homologues-alanine and glycine.Abbreviations GABA -aminobutyric acid - PES potential energy surface - RHF Roothaan-Hartree-Fock - SCF self consistent field     

Overexpression of γ－aminobutyric acid transporter subtype I leads to susceptibility to kainic acid－induced seizure in transgenic mice

Gamma-aminobutyric acid (GABA) is the principal inhibitory neurotransmitter, and the GABAergic synaptic transmission is normally terminated by the rapid uptake through GABA transporters. With transgenic mice ubiquitously overexpressing GABA transporter subtype I (GAT1), the present study explored the pathophysiological role of GAT1 in epileptogenesis. Though displaying no spontaneous seizure activity, these mice exhibit altered electroencephalographic patterns and increased susceptibility to seizure induced by kainic acid. In addition, the GABA(A) receptor and glutamate transporters are up-regulated in transgenic mice, which perhaps reflects a compensatory or corrective change to the elevated level of GAT1. These preliminary findings support the hypothesis that excitatory and inhibitory neurotransmission, and seizure susceptibility can be altered by neurotransmitter transporters.     

α-Methylene-γ-aminobutyric acid from Mycena pura

α-Methylene-γ-aminobutyric acid was isolated and characterized from fruit bodies of Mycena pura. It was the decarboxylation product of l-γ-methyleneglutamic acid by l-glutamic acid decarboxylase.     

Involvement of sialic acid in the regulation of γ--aminobutyric acid uptake activity of γ-aminobutyric acid transporter 1

The γ-aminobutyric acid (GABA) transporters (GATs) have long been recognized for their key role in the uptake of neurotransmitters. The GAT1 belongs to the family of Na(+)- and Cl(-)-coupled transport proteins, which possess 12 putative transmembrane (TM) domains and three N-glycosylation sites on the extracellular loop between TM domains 3 and 4. Previously, we demonstrated that terminal trimming of N-glycans is important for the GABA uptake activity of GAT1. In this work, we examined the effect of deficiency, removal or oxidation of surface sialic acid residues on GABA uptake activity to investigate their role in the GABA uptake of GAT1. We found that the reduced concentration of sialic acid on N-glycans was paralleled by a decreased GABA uptake activity of GAT1 in Chinese hamster ovary (CHO) Lec3 cells (mutant defective in sialic acid biosynthesis) in comparison to CHO cells. Likewise, either enzymatic removal or chemical oxidation of terminal sialic acids using sialidase or sodium periodate, respectively, resulted in a strong reduction in GAT1 activity. Kinetic analysis revealed that deficiency, removal or oxidation of terminal sialic acids did not affect the K(m) GABA values. However, deficiency and removal of terminal sialic acids of GAT1 reduced the V(max) GABA values with a reduced apparent affinity for extracellular Na(+). Oxidation of cell surface sialic acids also strongly reduced V(max) without affecting both affinities of GAT1 for GABA and Na(+), respectively. These results demonstrated for the first time that the terminal sialic acid of N-linked oligosaccharides of GAT1 plays a crucial role in the GABA transport process.     

Involvement of γ-aminobutyric acid transporter 2 in the hepatic uptake of taurine in rats

Taurine is essential for the hepatic synthesis of bile salts and, although taurine is synthesized mainly in pericentral hepatocytes, taurine and taurine-conjugated bile acids are abundant in periportal hepatocytes. One possible explanation for this discrepancy is that the active supply of taurine to hepatocytes from the blood stream is a key regulatory factor. The purpose of the present study is to investigate and identify the transporter responsible for taurine uptake by periportal hepatocytes. An in vivo bolus injection of [(3)H]taurine into the rat portal vein demonstrated that 25% of the injected [(3)H]taurine was taken up by the liver on a single pass. The in vivo uptake was significantly inhibited by GABA, taurine, β-alanine, and nipecotic acid, a GABA transporter (GAT) inhibitor, each at a concentration of 10 mM. The characteristics of Na(+)- and Cl(-)-dependent [(3)H]taurine uptake by freshly isolated rat hepatocytes were consistent with those of GAT2 (solute carrier SLC6A13). Indeed, the K(m) value of the saturable uptake (594 μM) was close to that of mouse SLC6A13-mediated taurine transport. Although GABA, taurine, and β-alanine inhibited the [(3)H]taurine uptake by > 50%, each at a concentration of 10 mM, GABA caused a marked inhibition with an IC(50) value of 95 μM. The [(3)H]taurine uptake exhibited a significant reduction when the GAT2 gene was silenced. Immunohistochemical analysis showed that GAT2 was localized on the sinusoidal membrane of the hepatocytes predominantly in the periportal region. These results suggest that GAT2 is responsible for taurine transport from the circulating blood to hepatocytes predominantly in the periportal region.     

Synergistic effect of phosphatidylserine with γ-aminobutyric acid in antagonizing the isoniazid-induced convulsions in mice

The influence of phosphatidylserine (PS) on the isoniazid-induced convulsions has been studied in mice. Sonicated dispersions of this phospholipid given intravenously do not show anticonvulsant activity but they do so when -aminobutyric acid (GABA) is simultaneously injected. GABA alone is inactive. The synergism between PS and GABA is influenced by the structure of the phospholipid liposomes. In contrast to multilamellar vesicles, oligolamellar vesicles are active. Under these conditions the effect shows head group specificity, in that the neutral phosphatidylcholine (PC) or the acidic phosphatidylinositol (PI) are inactive, either in the presence or in the absence of GABA. Lysophosphatidylserine (lysoPS), the deacylated PS derivative, shows increased efficacy as an isoniazid antagonist in the presence of GABA, and has anticonvulsant activity also in the absence of GABA. Other lysophospholipids are inactive. It is suggested that PS, after its metabolic conversion to lysoPS, enhances the anticonvulsant effect of GABA.     

γ-aminobutyric acid in the central nervous system of metamorphosing and mature Manduca sexta

We have begun to examine the factors controlling the accumulation of the neurotransmitter γ-aminobutyric acid (GABA) in the central nervous system (CNS) of the sphinx moth Manduca sexta. Analysis of soluble amino acids in CNS structures from mature moths outlines the regional distribution of GABA. Analysis of amino acids in the antennal lobes (the primary olfactory centres) of Manduca during metamorphosis reveals that GABA accumulates gradually and continuously through most of adult development until eclosion; within 18 hr after eclosion, levels of GABA abruptly increase 27–50%. The activity of the biosynthetic enzyme glutamic acid decarboxylase (EC 4.1.1.15), assayed in extracts of antennal lobes from developing moths, does not change after eclosion. Extracts of hemolymph from mature moths contain low levels of glutamate ( <0.2 mM) and higher levels of certain other amino acids such as serine, glutamine and proline. The concentration of proline in hemolymph increases up to 2-fold after eclosion. Glutamate, glutamine and proline are interconvertible in the CNS, and each can serve as precursor for GABA synthesis both in vivo and in vitro. The efficiency of the precursor role in vitro is similar for each amino acid, as estimated from the ratio of the specific radioactivities of GABA and glutamic acid in the ganglion derived from each precursor. Exogenous proline and glutamine can equilibrate rapidly with the ganglionic pools of the same amino acids while glutamic acid is relatively excluded. Taken together, the findings of this study show that proline and glutamine may contribute substantially to synthesis of GABA in the CNS of M. sexta.     

Glutamic acid decarboxylase of embryonic avian retina cells in culture: Regulation byγ-aminobutyric acid (GABA)

1. Retina-cell aggregate cultures expressed glutamate decarboxylase activity (L-glutamate 1-carboxylase; EC 4.1.1.15) as a function of culture differentiation. 2. Glutamic acid decarboxylase (GAD) activity was low in the initial phases of culture and increased eight-fold until culture day 7, remaining high up to day 13 (last stage studied). 3. The addition of GABA to the culture medium 24 h after cell seeding almost totally prevented the expression of GAD activity. 4. In association with decreased enzyme activity, aggregates exposed to GABA did not display immunoreactivity for GAD, suggesting that GAD molecules were either lost from GABAergic neurons or significantly altered with GABA treatment. 5. Control, untreated aggregates showed intense GAD immunoreactivity in neurons. Positive cell bodies were characterized by a thin rim of labeled cytoplasm with thickest labeling at the emergence of the main neurite. 6. Heavily labeled patches were also observed throughout the aggregates, possibly reflecting regions enriched in neurites. 7. The GABA-mediated reduction of GAD immunoreactivity was a reversible phenomenon and could be prevented by picrotoxin.     

γ—Aminobutyric acid transporter （GAT1） overexpression in mouse affects the testicular morphology

γ-Aminobutyric acid and GABAergic receptors were previously reported to be distributed in reproductive systems besides CNS and predicted to participate in the modulation of testicular function.γ-Aminobutyric acid transporter was implicated to be involved in this process.However,the potential role of γ-aminobutyric transporter in testis has not been explored.In this study,we investigated the existence of mouse γ-aminobutyric acid transporter subtype I (mGAT1) in testis.Wild-type and transgenic mice,which overexpressing mGAT1 in a variety of tissues,especially in testis,were primarily studied to approach the profile of mGAT1 in testis.Mice with overexpressed mGAT1 develop normally but with reduced mass and size of testis as compared with wild-type.Testicular morphology of transgenic mice exhibited overt abnormalities including focal damage of the spermatogenic epithelium accompanied by capillaries proliferation and increased diameter of seminiferous tubules lumen.Reduced number of spermatids was also found in some seminiferous tubules.Our results clearly demonstrate the presence of GAT1 in mouse testis and imply that GAT1 is possibly involved in testicular function.     

Autoradiographic localization of 3H-γ-aminobutyric acid uptake in the lamina ganglionaris of Musca and Drosophila

Summary The uptake of ³H-GABA in the visual system of half-head preparations of Musca and Drosophila was studied by means of light and electron microscope autoradiography. Of all three ganglia, only the first synaptic region, the lamina ganglionaris, showed accumulation of radioactive grains, and there a preferential glial uptake could be found. Under normal light conditions at incubation (constant light flux of 100 Lux) the maximum of radio-activity was found in the marginal glia cells. Increasing the time of incubation produced also an increase in the number of grains per surface unit in the marginal glia cells. After changing the light intensity during incubation, quantitative modifications of the distribution of radio-activity were observed: incubating with stroboscopic illumination, the number of grains diminished in the marginal glia cells and remained constant in the epithelial cells; incubated in darkness, the epithelial cells became more intensely labelled whilst the number of grains decreased in the marginal cells.The possibility is discussed that the receptor axons 1–6 are the neurological elements of the lamina which use GABA as a transmitter. This hypothesis is lent some support from results of similar experiments with neurological mutants of Drosophila. In opm 18 there was a delayed uptake of ³H-GABA whereas in opm 3 and ebony the results were comparable to those found in Musca incubated in darkness. Behavioral studies on these mutants have demonstrated a defect, most probably related to the receptor system 1–6.     

Role of uptake inγ-aminobutyric acid (GABA)-mediated responses in guinea pig hippocampal neurons

Intracellular recordings were obtained from hippocampal pyramidal neurons maintained in vitro. Measurements were made of the conductance change induced by iontophoretically applied gamma-aminobutyric acid (GABA) and, using voltage-clamp techniques, of inhibitory postsynaptic currents resulting from activation of inhibitory pathways. Analysis of GABA iontophoretic charge-response curves indicated that there was considerable variation among neurons with respect to the slope of this relation. The placement of the GABA-containing pipette did not appear to be responsible for the observed variation, since vertical repositioning of the pipette did not alter the slope of the charge-response relationship. Steady iontophoresis of GABA from one barrel of a double-barreled pipette markedly affected the charge-response relation obtained when short pulses were applied to the other barrel. The curve was shifted to the left, and the slope was decreased. Concomitantly, the enhanced GABA-induced responses were prolonged. Similar alterations in GABA responsiveness were observed when the uptake blocker, nipecotic acid, was iontophoretically applied. Furthermore, bath application of saline containing a reduced sodium concentration (25% of control) also produced a prolongation of GABA-mediated responses. Under voltage clamp, inhibitory postsynaptic currents were observed to have biphasic decays. The initial, fast decay was prolonged by an average of 18% by nipecotic acid, whereas the later, slow phase was prolonged by 23%. The results of these studies support the hypothesis that a saturable GABA uptake system is responsible for the observed variation in the charge-response curves and, in turn, underlies the apparent sensitizing effect of excess GABA application. The results also suggest that a reduction of transmitter uptake affects the time course of inhibitory postsynaptic currents in the hippocampus.     

Interactions ofγ-aminobutyric acid (GABA), pentobarbital,and homopantothenic acid (HOPA) on internally perfused frog sensory neurons

Augmentatory actions among Cl- currents (ICl) induced by gamma-aminobutyric acid (GABA), pentobarbital (PB), and homopantothenic acid (HOPA) were investigated in isolated frog sensory neurons after suppression of Na+, K+, and Ca2+ currents using a suction pipette technique which combines internal perfusion with voltage clamp. GABA-sensitive neurons responded to both PB and HOPA, and the responses behaved as a simple Cl- electrode and reversed at the Cl- equilibrium potential (ECl). The dose-response curve for GABA-induced Cl- conductance was sigmoidal with the GABA concentration producing a half-maximum response (4.2 X 10(-5) M). Both GABA and HOPA dose-response curves shifted to the left in the presence of PB, though the facilitatory action of PB on GABA- and HOPA-induced ICl was more effective in the former. There was a significant facilitatory interaction between GABA- and HOPA-induced ICl. It is concluded that HOPA affects the GABA-GABA or PB-PB receptor interactions.     

Oral treatment with γ-aminobutyric acid improves glucose tolerance and insulin sensitivity by inhibiting inflammation in high fat diet-fed mice

Adipocyte and β-cell dysfunction and macrophage-related chronic inflammation are critical for the development of obesity-related insulin resistance and type 2 diabetes mellitus (T2DM), which can be negatively regulated by Tregs. Our previous studies and those of others have shown that activation of γ-aminobutyric acid (GABA) receptors inhibits inflammation in mice. However, whether GABA could modulate high fat diet (HFD)-induced obesity, glucose intolerance and insulin resistance has not been explored. Here, we show that although oral treatment with GABA does not affect water and food consumption it inhibits the HFD-induced gain in body weights in C57BL/6 mice. Furthermore, oral treatment with GABA significantly reduced the concentrations of fasting blood glucose, and improved glucose tolerance and insulin sensitivity in the HFD-fed mice. More importantly, after the onset of obesity and T2DM, oral treatment with GABA inhibited the continual HFD-induced gain in body weights, reduced the concentrations of fasting blood glucose and improved glucose tolerance and insulin sensitivity in mice. In addition, oral treatment with GABA reduced the epididymal fat mass, adipocyte size, and the frequency of macrophage infiltrates in the adipose tissues of HFD-fed mice. Notably, oral treatment with GABA significantly increased the frequency of CD4(+)Foxp3(+) Tregs in mice. Collectively, our data indicated that activation of peripheral GABA receptors inhibited the HFD-induced glucose intolerance, insulin resistance, and obesity by inhibiting obesity-related inflammation and up-regulating Treg responses in vivo. Given that GABA is safe for human consumption, activators of GABA receptors may be valuable for the prevention of obesity and intervention of T2DM in the clinic.     

Effects of repetitive transcranial magnetic stimulation on ER stress-related genes and glutamate, γ-aminobutyric acid and glycine transporter genes in mouse brain

Repetitive transcranial magnetic stimulation (rTMS) is an emerging therapy for the treatment of psychiatric disorders. However, the mechanisms underlying the therapeutic effects of rTMS are still unclear, limiting its optimisation. Lasting effects suggest changes in disease-related genes, so we conducted gene chip and qRT-PCR analyses of genes associated with psychiatric diseases in the mouse brain at various times following 1, 20, 30 or 40 days of rTMS. Many genes were differentially expressed in the rTMS-treated mouse brain compared to sham controls, including genes encoding neurotransmitter transporters (upregulation of EAAT4, GLAST, GLT-1, GAT2, GAT4, GLYT1 and GLYT2), and endoplasmic reticulum (ER)-stress proteins (downregulation of IRE1α, IRE1β, and XBP1, upregulation of ATF6 and GRP78/Bip). Expression changes in many of these genes were also observed 10 days after the last rTMS treatment. In PC12 cells, rTMS upregulated GRP78/Bip mRNA and enhanced resistance against H₂O₂ stress. These results suggest that rTMS differentially modulates multiple genes associated with psychiatric and neurodegenerative disorders. Sustained changes in the expression of these genes may underlie the therapeutic efficacy of chronic rTMS.